Potential role of Citrus bergamia flower essential oil against oral pathogens

Background Oral bacterial infections are difficult to treat due to emergence of resistance against antibiotic therapy. Essential oils are considered emerging alternate therapy against bacterial infections and biofilms. We investigated Citrus bergemia flower essential oil against oral pathogens. Methods The essential oil was analsyed using Gas Chromatography(GC–MS), in silico investigations, antioxidant, antimicrobial, antibiofilm and antiquorum sensing assays. Results Gas Chromatography analysis confirmed presence of 17 compounds including 1,6-Octadien-3-ol,3,7-dimethyl, 48.17%), l-limonene (22.03%) and p-menth-1-ol, 8-ol (7.31%) as major components. In silico analysis showed compliance of all tested major components with Lipinski’s rule, Bioavailability and antimicrobial activity using PASS (prediction of activity spectrum of substances). Molecular docking with transcriptional regulators 3QP5, 5OE3, 4B2O and 3Q3D revealed strong interaction of all tested compounds except 1,6-Octadien-3-ol,3,7-dimethyl. All tested compounds presented significant inhibition of DPPH (2,2-diphenyl-1-picrylhydrazyl) (IC50 0.65 mg/mL), H2O2 (hydrogen peroxide) (63.5%) and high FRAP (ferrous reducing antioxidant power) value (239.01 µg). In antimicrobial screening a significant activity (MIC 0.125 mg/mL) against Bacillus paramycoides and Bacillus chungangensis was observed. Likewise a strong antibiofilm (52.1 – 69.5%) and anti-QS (quorum sensing) (4–16 mm) activity was recorded in a dose dependent manner. Conclusion It was therefore concluded that C. bergemia essential oil posess strong antioxidant, antimicrobial and antibiofilm activities against tested oral pathogens. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-024-04457-7.


Background
The Food and Agricultural Organization (FAO) enlists citrus as one of the most important crops with reference to annual production across the globe [1].Citrus fruits are being used since ancient times for management of several health related problems including scurvy, common cold, menstrual irregularities, myocardial infarction, coronary artery disease and high blood cholesterol due to strong antioxidant potential [2,3].Citrus fruits and peel are considered as rich source of diverse flavonoids, polyphenolic compounds, carboxylic acids, vitamin C and amino acids [4,5].Amongst all Citrus bergamia (Rutaceae, C. bergemia) has gained great attraction from researchers because of its peculiar health benefits [6].C bergemia is native to Italy and Spain however it is cultivated throughout the world [7].The geographical and botanical origins of this particular fruit are still uncertain [8].
Bergamot contains essential oils in peel and flowers that has largely been used in medical, food, cosmetics, perfumes, and confectionary industry [3].The essential oils (EOs) are complex mixtures of chemical compounds, each having a unique intense odor, and are located in different parts of the plants including seeds, fruits, stems, leaves, roots, and flowers [9].The EOs are the secondary metabolites formed as a result of plant defensive mechanisms through the involvement of enzymes [10].It has been emphasized that EOs are the final terpenoid products produced in plants as a result of enzymes like terpene synthase [11].Numerous studies have reported the therapeutic potential of the EOs including antimicrobial, anticancer, and antioxidant properties.Being an antimicrobial moiety, EOs are also used to preserve food items [12].Bergemia essential oil (BEO) is traditionally used for the treatment of parasitic infections, sore throat, tonsillitis, wound healing, hyperhidrosis, vaginal pruritis, leucorrhoea, skin and mouth infections, gonococcal infections, urinary tract and respiratory tract infections [13].
Oral complications including dental abscess, gingivitis, dental caries and periodontitis are mainly due to oral bacterial infections [14,15].These infections are main reason for global health burden ($442 billion) [16] and considered serious since these can lead to cancer and death in worst conditions [17].It has been estimated that a huge population (3.5 billion) of the world is currently effected by oral diseases untreated dental caries is one of the most common non-communicable disease [14].The treatment failures and development of antibiotic resistance [18] has and the importance of natural origin antimicrobials motivated us to explore the effect of the bergemia flower essential oil (BFEO) effect against oral microorganisms.The core aim of the study was to investigate BFEO against oral pathogens using in silico and in vitro models.

Plant material and isolation of essential oil
The fresh flowers of Citrus bergemia were collected with consent from farm house in Paharpur area (District D.I.Khan, KPK, Pakistan) and authenticated by Dr. Zain Ul Abedene (Taxonomist in Institute of Biosciences, Gomal University D.I.Khan, Pakistan).The collected flowers (50 g) were stored at -40 °C for essential oil extraction in clevenger-type apparatus to isolated essential oil using hydro-distillation.The anhydrous sodium sulphate (anhydrous) was used for drying of collected essential oil and removal of water.Finally, essential oil was stored at 4 °C.

Microbial strains and growth media
The clinical strains were isolated from dental plaque of female diabetic patients with the help of Dentist.Strains were transferred to lab and purifications, isolation of strains was performed using differential media including EMB (eosin methylene blue) agar, Mackonkey agar and congo red agar.The strains were identified by 16S rRNA as Bacillus chungangensis, Bacillus paramycoides, Bacillus chungangensis, Paenibacillus dendritiformis, Staphylococos aureus and Staphylococus epidermidis.Standard bacterial strains were E.coli (ATCC 25922), Staphylococcus aureus (ATCC 33862), Klebsiella pneumoniae (ATCC BAA-1705).Biomarker strains included Chromobacterium violaceum (DSM 30191).Growth and differential media used during the investigation including Lauria bertani (LB), BHIA (brain heart infusion agar), tryptic soya broth (TSB) were purchased from Hi Media (India) while Mackonkey agar and eosin methylene blue agar was purchased from (Oxoid, UK).

GC-MS analysis
Essential oil component analysis was accomplished by means of GCMS (Shimadzu GC 2010, Japan) having installed, auto sampler (AOC-20i autosampler) and suitable capillary column (dimensions 30 m × 0.25 mm id, 0.25 μm film thickness, a DB-5 MS column).System oven temperature was set (initially at 45-90 °C) with a rise rate of 2 °C (per min), then increase from 91 to 240 °C with a rise rate of 3 °C (per min).Finally achieved temperature (240 °C) was set constant for 5 min.The temperature of injector (240 °C) and detector (280 °C) was kept constant at set temperatures.For loading the sample, an aliquot of essential oil was (0.5 μL) was injected and Helium (1 ml/ min) was used as a carrier gas.GCMS component analysis and identification was accomplished on a GCMS-QP 2010 Plus (Shimadzu, Japan) system operating in EI mode (electron ionization mode) at 70 eV.Mass units were monitored from 35 to 500 AMU.The NIST mass spectral library and compound mix (including limonene, carvacrol, thymol, α-pinene, β-pinene, β-myrcene and sabinene) was used for identification of compounds [19].

Molecular docking
For Molecular docking studies, the X-ray crystallographic structures of the transcriptional regulators LasR (2UV0), PqsE (2Q0J) [25] and quorum sensing regulators CviR (3QP5) [26] were obtained from the Protein Data Bank (PDB).The active site dimensions for each protein were recorded by using their co-crystallized ligands respectively.Then, the water molecules and co-crystallized ligand were removed and hydrogen atoms and charges were added.The SDF format for 3D structures of all the phyto-constituents were downloaded from Pubchem database and PDB files were generated in Accelrys DS Visualizer 2.0 (Accelrys Software Inc., 2012).The molecular docking was performed using Lamarckian Genetic Algorithm embedded in AutoDock v 4.2.[27].A total number of 63 different poses were generated and clustered according to their RMSD values.Each cluster was carefully visualized in Discovery Studio Visualizer [28] and putative binding modes were selected accordingly.Best docked structures based on the binding energy scores (ΔG) were chosen for further analyses.The hydrogen bonding and hydrophobic interactions between ligand and protein were calculated by Accelrys DS Visualizer 2.0 [29].

Antioxidant activities
Hydrogen peroxide (H 2 O 2 ) assay A modified method was employed for determination of H 2 O 2 based antioxidant activity [30].Concisely, H 2 O 2 stock solution (2 mM) was prepared followed by mixing (600 µL) with test sample (400 µL).The reaction solution was vortexed and absorbance was measured after 10 min at 230 nm.As reference standard, ascorbic acid was used and results were expressed as below where A 0 is absorbance of sample and A1 was absorbance of sample.

DPPH Assay
The antioxidant activity of samples was determined by using modified procedure [31].Briefly, DPPH (freshly prepared) solution in methanol (0.5 mL; 0.1 mM) was combined with plant extract (0.5 mL, several concentrations) and placed 30 min (In dark place to avoid light effect).Later absorbance was observed at 517 nm.As reference standard, ascorbic acid was used.Results were expressed as below; A linear plot was used to calculate IC 50 values of test samples.
FRAP assay Ferric ion reducing power of tested sample was determined using modified method [32].Concisely, fresh FRAP reagent was set by adding TPTZ (10 mM prepared in 40 mM HCL) to acetate buffer (300 mM), and ferric chloride (20 mM) in specified ratio (10:1:1).Tested sample (100 µL) was reacted with FRAP reagent (3 mL) and placed in dark place (30 min).Afterwards, absorbance was recorded (at 593 nm) in UV-Vis spectrophotometer.The ferrous reducing capacity was determined by FeSO 4 standard curve and results were expressed as µg equivalents.

Antimicrobial activities
Determination of MIC The isolated compounds were assessed for antimicrobial activities using modified method [33] with slight modifications.In the MIC assay, the 96 microwell plates were loaded with 50 µL of the overnight-grown bacterial strain (1.5 × 10 7 CFU/mL), followed by addition of 50 µL of test sample (various dilutions).The plates were incubated at 37 °C for 24 h.On the next day, 40 µL of resazurin solution (0.015%) was added to each well followed by incubation at 37 a °C for further 60 min.Colorimetric readings were recorded using 96-microplate reader (Hippo MPP-96, Biosan).For MBC values, bacterial suspensions (10 μL) from the MIC microwell were relocated to already prepared agar plates (Muller Hinton) and incubated for 24 h.Afterwards, bacterial growth was recorded on the agar plates.All samples were loaded in triplicate.Ciprofloxacin was used as positive control.

% Inhibition
Antibiofilm activity The biofilm formation assay was performed using 12-well polystyrene plates with a slightly modified method [34].Briefly, the bacterial strain was inoculated in TSB medium (280 μL) at an initial turbidity of 0.5 at 600 nm (0.5 McFarland).And allowed to incubate for 24 h to produce biofilm.Afterwards 100 μL of test compound (0.01-3 mg/mL) was added to the bacterial culture followed by incubation at 37 °C for further 24 h.Cell growth in the plates was measured at 592 nm.For quantification, the biofilms in the 12-well plates were stained using crystal violet.Afterwards 95% ethanol was added to the stained cells and absorbance was recorded at 592 nm to quantify total biofilm formation.
The % inhibition was calculated using following formula Antiquorum sensing The quorum sensing inhibition potential of isolated compounds was evaluated by a standard procedure [34] with slight modifications.An overnight culture of Chromobacterium.violaceum (1/100 ratio) was streaked onto LB agar in Petri dishes.Sterilized filter paper discs (6 mm) were prepared and placed on the top of BHIA (Brain Heart Infusion agar) seeded with indicator strain (C.violaceum).Then 15µL test compound (0.01-3 mg/mL) was applied on each disc and allowed to dry for 30 min.Afterwards, the assay plates were incubated at 30 °C for 3 days.Ciprofloxacin was used as standard drug.Finally, results were recorded by measuring the zone of inhibition around each disc.

Violacin inhibition assay
A modified method [34] was adopted for violacein inhibition assay.A 24 h old culture (200 μL of C. violaceum (OD = 0.4 OD at 600 nm) was loaded to sterilized microtiter plates containing various concentrations of compounds (1-4 mg/mL).The plates were incubated at 30 °C for 24 h and witnessed for the decrease in violacin pigment production by taking absorbance at 585 nm.The percentage inhibition was calculated by following the formula:

Statistical analysis
All biological activity experiments were performed in three independent experiments data was expressed as ± SD.One way ANOVA followed by post-hoc Tukey test with p < 0.05.

Drug likeness, bioavailability and PASS analysis
Major identified components of C. bergemia were checked for drug likeness using Lipinski's rule of five.It was evident that all tested compounds (except β-pinene) showed a compliance with lipinski's rule and therefore were considered as suitable candidates for further  analysis (Table 2).Since Log p value of β-pinene (Log p 4.14) showed a slight deviation from set value (Log p < 5), a violation was observed.Further, drug likeness scores of l-limonene, p-menth-1 en-8-ol, β-myrcene and β-pinene presented a slight deviation from standard value (+ 1 to -1) (Table 2, Fig. 3).The pharmacokinetic parameters were evaluated by using SWISS ADME (Table 3).It was evident from boiled egg model that all tested components were able to cross blood brain barrier (BBB) except aromadendrine.Whereas Bioavailability radar showed that limonene, β-myrcene had low absorbtion from GIT.Further, all parameter (insaturation, polarity, Molecular weight, insolubility, flexibility and lipophilicity) of bioavailability radar were with permissible limits for tested compounds except for aromadendrine with a slight increase in case of in saturation (Fig. 4).PASS analysis revealed that all tested compounds had a potential to interact with bacterial cells (0.4973 -0.6049 confidence interval) (Table 4).Therefore these potential drug candidates were further processed for selective molecular docking analysis.

Antimicrobial screening
The isolated clinical strains were screened for their antibiograms and it was evident that among all tested antibiotics only Paenibacillus dendritiformis and Bacillus paramycoides and Bacillus chungangensis were susceptible to imipenem whereas a borderline sensitivity was recorded against Bacillus chungangensis (Table 7).Likewise Paenibacillus dendritiformis was sensitive to ciprofloxacin, whereas a border line sensitivity was seen against both isolates of Bacillus chungangensis.All other antimicrobial agents were resistant to isolated strains (Table 7).The C. bergemia essential oil was analsyed against isolated strains and a moderate inhibition was observed.Amongst tested strains, highest activity (MIC 0.125 mg/ mL) was recorded against Bacillus paramycoides and Bacillus chungangensis (Table 8).In comparative analysis, C. bergemia essential was analsyed against standard strains (ATCC) and a significant inhibition was noticed against all tested strains (0.0312-0.0625 mg/mL) (Table 9), that was an indication of prominent activity against resistant strains.

Antibiofilm and anti Q.S activities
Our preliminary investigations (congo red agar) confirmed that all clinical strains were biofilm producers, and thus antibiofilm activities were performed.A significant inhibition of biofilm produced by all tested strains was recorded (Fig. 8) with highest inhibition (69.8%) seen in case of Paenibacillus dendritiformis.Likewise, C. bergemia essential oil showed significant inhibition of C. vioalceum at diverse concentration (Fig. 9) that indicated significant antiquorum sensing potential.This was further evident from violacein inhibition assay where a significant inhibition of violacine inhibition was noticed (Fig. 10).Thus the antibiofilm activities of C. bergemia can be attributed to anti QS inhibition potential.

Discussions
Citrus bergemia is an important medicinal plant, whose several parts has usage in traditional medicinal systems including Italian, Greece and Chinese [35].Generally, essential oil from peel of C. bergemia is known for antiinflammatory, antibacterial anticancer, antidiabetic antiviral properties [36,37].We explored flower essential of this plant against oral pathogens, since prevalence rate of such infections is too high [38] and existing therapies (antibiotics) have become resistant.The prevalence rate of such infections is too high in developing countries [39], thus there exists a great potential for new alternative treatments.To best of our knowledge, this is first report exploring potential of C. bergemia against oral pathogens.
Online computational tools have become a tool of prime importance in drug discovery these days.These tool are based on several algorithms, that enable simulation with reference to targets, and thus provide a prediction with high probability [43].We investigated major components for their druglikeness, bioavailability and possible antimicrobial targets.As explained earlier, all major components followed lipinski's rule of five however as light deviations from drug likeness scores were noticed.However, they were minor and may have a little effect on drug bioavailability.Never the less, in limonene, β-myrcin and β-pinene the number of H-bond donors and acceptors were zero, that indicate a limited or no bond formation with target amino acids, thus limiting the biological activity and polarity imbalance that may effect permeability and drug solubility [44].The   pharmacokinetic spectrum analysis revealed that except aromdendrene, all drugs may cross the blood brain barrier and therefore may have effect on CNS.This could possibly be reason that essential oils are effectively used in aromatherapy [45].Likewise, all parameters of tested compounds followed the cut points of bioavailability radar and therefore regarded as molecules with agreeable bioavailability.cytochrome p450 enzyme in liver are very important for drug metabolism and ADMET analysis indicated that most of tested drug molecules were neither substrate nor enzymes for such enzymes, that show lower metabolism in liver [46].Once the initial drug likeness and bioavailability parameters checking, we investigated possible antimicrobial potential using PASS analysis.The prediction of activity spectrum of substances is an important tool that indicates probability of possible biological activity [24] that was antimicrobial potential in this case.A high confidence interval in PASS results indicated a good possible antimicrobial activity.
Oxidative stress is a key marker that leads to several diseases including bacterial infections [47].Several investigations have shown higher oxidative stress in several bacterial infections, that is possibly due to altered metabolic pathways and generation of reactive oxidation   products (ROS) during bacterial infections [48].C. bergemia essential oil showed high phenolic contents and it was obvious that it may have strong antioxidant activities [49], as evident from DPPH, H 2 O 2 and FRAP assays.The essential oils are mainly consisted up of monoterpenoids that participate in a oxidative chemical reaction by react through H atom and inhibition of free radicals chain in reaction [50].Further compounds with strong antioxidant activity may have strong antimicrobial properties [51].
The C. bergemia essential oil presented significant inhibition of resistant strains, that showed potential of this essential oil against oral pathogens.Interestingly the MIC of standard strains against C. bergemia was several  fold higher.The strong antimicrobial activity was attributed to presence of monoterpenes, that may interacted synergistically.Studies have shown that monoterpenoids due to their lipophilic nature are mainly partitioned from an aqueous phase into bacterial membrane structures [52].This partitioning effect leads to increase in membrane permeability, destruction of membrane bound structures, interference with ion transport and bacterial cell membrane expansion [53].Our experiment using congo red assay confirmed that all tested strains were biofilm producers, and thus we investigated C. bergemia essential oil for their potential biofilm activities.It was observed that C. bergemia showed a significant inhibition of biofilms produced by tested strains by block cell-cell signaling mechanism (quorum sensing) in a dose dependent manner.Bacterial biofilms are includes a matrix of extracellular polymeric substances, that limit the activity of antimicrobial agents to kill bacteria and their permeability to reach target site [54], that leads to antimicrobial resistance.Investigators have shown that monoterpenoids mainly inhibit biofilm formation at early stage by inhibiting formation of flagella [55] blocking biosynthesis of poly-n-acetylglucosamine polymers biosynthesis, that are major elements of bacterial biofilm and quorum sensing [56].

Conclusion
Oral bacterial infections are important health concern that may lead to development of serious complications of oral cavity.Essential oils are important remedies for ailment of bacterial infections including oral pathogens.We concluded that C. bergemia flower essential oil posess significant anti-microbial, antibiofilm and anti Qs activities against oral pathogens including Bacillus chungangensis, Bacillus paramycoides and Paenibacillus dendriformis that can be due strong antioxidant potential.The seasonal variations do effect essential oil concentrations and therefore biological activities too, thus future studies are required to investigate essential yield, collection time and biological activities.

Fig. 2
Fig. 2 Structure of major components detected in C. bergemia essential oil

Fig. 9
Fig. 9 Anti Quorum sensing activities of C. Bergemia essential oil

Table 1
GC-MS Profile of Citrus bergemia flower essential oil

Table 3
Pharmacokinetic parameter of major components from C. bergemia using SWISS-ADME tool

Table 4
PASS of major components from C. bergemia using online tools (Ways2drugs)

Table 5
Docking score and interaction analysis of major components in C. bergemia

Table 6
Antioxidant activity of C. bergemia flower essential oil

Table 7
Antibiogram studies of oral pathogens against selected antimicrobial agents

Table 9
Determination of MIC against standard strainsStandard drug imipenem 44.4 µg/mL against Klebsiella pneumoniae, 22.4 µg/mL against all other strains values are means of triplicate determination (n = 3)